Regulation of L-type calcium channel inactivation

钙离子通过膜电压变化激活的钙通道进入大脑中的神经元，这一过程对从基因转录到膜兴奋性等一系列功能至关重要。事实上，钙作为信号分子的重要性决定了系统需要精确地调节钙进入的时间和程度。L类钙通道在大脑中广泛表达，并受钙调蛋白(CaM)的调节，CaM是一种通过钙依赖失活或钙依赖促进(CDF)过程调节CaV1钙内流的分子。最近的工作表明，Cav1介导的钙内流可以通过与通道C末端结合的辅助蛋白来显著增加，从而减少CDI。两个例子是结合到C末端以招募专门的激酶CaMKII的支架蛋白Densin，第二个例子是钙敏感蛋白calendrin，它物理上取代了Cav1通道上的CaM。我们有一种新型的持续5秒的Cav1.3通道易化(称为L-CDF)的初步数据，它跟随着一系列简短的电路活动的输入特征。此外，Cav1.3 L-CDF依赖于密度蛋白和CaMKII的表达。这一点很重要，因为这些蛋白中的每一种都在CA1海马体锥体细胞中表达，在短暂的突触输入后，钙通道活动的“延迟促进”增强了钙内流。我们假设辅助蛋白致密蛋白和钙树突蛋白是促进锥体细胞钙内流大量增加的延迟易化的基础。我们将应用分子生物学、生物化学和电生理学等技术，研究在模型系统和啮齿动物脑组织切片中表达的L型钙通道，以确定致密蛋白和钙树突蛋白在Cav1通道L-CDF产生中的作用。总之，这项工作代表了我们实现长期目标的第一步，即了解L类钙通道是如何调控内部钙离子和第二信使通路的激活的。Cav1通道在从大脑到肌肉和心脏的所有组织中的广泛表达将确保结果将立即对理解如何在多种环境下控制钙内流具有重要意义，从而促进人们对体内可兴奋细胞的功能如何受到这些蛋白质-蛋白质相互作用的调控的了解。

Calcium ions enter neurons in the brain through calcium channels activated by a change in membrane voltage, a process that is critical to functions ranging from gene transcription to membrane excitability. Indeed, the importance of calcium as a signaling molecule dictates the need for systems to precisely regulate the timing and extent of calcium entry. The “L-type” (Cav1) class of calcium channels is widely expressed in the brain and is regulated by calmodulin (CaM), a molecule that modifies Cav1 calcium influx through a process of “calcium-dependent inactivation” (CDI) or “calcium-dependent facilitation” (CDF). Recent work reveals that Cav1-mediated calcium influx can be substantially increased by accessory proteins that bind to the channel C terminus to reduce CDI. Two examples are the scaffolding protein densin that binds to the C terminus to recruit the specialized kinase CaMKII, and a second is the calcium sensitive protein caldendrin that physically displaces CaM on the Cav1 channel. We have preliminary data for a novel form of Cav1.3 channel facilitation that lasts for >5 seconds (termed L-CDF) following a brief train of input characteristic of circuit activity. Moreover, Cav1.3 L-CDF depends on expression of densin and CaMKII. This is important, as each of these proteins are expressed in CA1 hippocampal pyramidal cells, where a “delayed facilitation” of calcium channel activity after a brief train of synaptic input enhances calcium influx. We hypothesize that the accessory proteins densin and caldendrin underlie the delayed facilitation that promotes a large increase in calcium influx in pyramidal cells. We will apply techniques ranging from molecular biology to biochemistry to electrophysiology to study L-type calcium channels expressed in a model system and in slices of rodent brain tissue to determine the role of densin and caldendrin in producing Cav1 channel L-CDF. Together this work represents the first step of attaining our long term goal ofunderstanding how L-type calcium channels are regulated to controlinternal calcium and activation of second messenger pathways. The widespread expression of Cav1 channels in all tissues ranging from brain to muscle and heart will ensure that the results will be immediately important to understanding how calcium influx is controlled in multiple contexts, advancing knowledge of how the function of excitable cells in the body is regulated by these protein-protein interactions.